Optical detection methods are frequently employed in analytical instruments for sensitive and species-specific detection of chemical compounds. Optical detection is particularly well suited for analytical techniques such as high performance liquid chromatography (HPLC). HPLC has been used for many years as a means of separating, identifying, purifying and quantifying components of often-complex mixtures. HPLC is an important tool used by biotechnological, biomedical, and biochemical research as well as in the pharmaceutical, cosmetics, energy, food, and environmental industries.
Typically, the optical systems associated with HPLC detectors use a series of lenses and mirrors to collect and propagate light from a source through free space to a flow cell. Fluid containing analytes that have been separated by elution through a chromatographic column flows through this cell. Light not absorbed after passing through this cell strikes a light detector containing one or more photosensitive elements.
Propagation of light through free space has certain disadvantages. Specifically, for satisfactory operation, the mechanical stability of each optical component has to be extremely high to ensure the optical path length is faithfully maintained. In addition, beam tubes often have to be used to alleviate the susceptibility to air currents, which can change the local refractive index and slightly bend light rays, contributing uncertainties (noise) in sensitive measurements. Lastly, losses of light intensity occur due to Fresnel reflections at each air-glass interface due to the sudden change in refractive index.
These disadvantages can be largely avoided by propagating light through optical fibers. Using an optical fiber to transmit light has additional advantages. Doing so minimizes the use of optical elements such as collimating lenses and reflectors, particularly when path lengths are extended. Further, optical fibers are flexible, allowing the primary light source to be located in places not feasible if the light were being propagated through free space. Lastly, optical fibers can transmit light with little or no loss of intensity, even over extended distances.
Because the location of the primary light source relative to the optical detector is less important when using optical fiber and because a single primary light source can illuminate multiple optical fibers simultaneously, a single source of light can be directed to a number of optical detectors simultaneously via multiple optical fibers. This will be useful in parallel HPLC systems, which are currently on the verge of introduction to the marketplace and will further increase sample processing speed in analytical instrumentation.
One example of a prior art device where a single primary light source illuminates multiple optical fibers is the Waters Corporation Model 2488 Multichannel Absorbance Detector. According to its Operator's Guide, Waters 2488 Multichannel Absorbance Detector with MassLynx Control, Operator's Guide, 71500248802 Revision A, Waters Corporation, Milford, Mass. (2001), the detector uses a deuterium lamp followed by a combination of mirrors and a grating to select light within a single wavelength band and to focus this light directly onto the input ends of eight optical fibers. The input ends of the eight short optical fibers are arranged in two parallel rows of four, one row offset from the other by half of a fiber diameter for closer packing. The eight fibers transmit the wavelength-selected light that is then focused through respective sample detection cells and onto associated detection photodiodes. This system is a hybrid one, combining optical fibers with a number of free space optical components. There are also a number of other commercially available optical detection systems incorporating optical fibers into their design.
As with many other single wavelength instruments, a beam splitter located between the optical fibers and the deuterium lamp directs some of the single wavelength light into a reference photodiode. This allows the output of the deuterium lamp to be monitored independent of absorption by any sample. In theory, when analyzing data from the sample detection cells, compensation can then be made for any fluctuations in the intensity of the light from the lamp.
Incoherent light sources, such as a deuterium lamp, are often treated theoretically as point sources, i.e. filling no volume. In reality they do have a finite spatial extent and the intensity and spectral distribution of the light emanating from different areas of the incoherent light source volume can vary strongly. For this reason, if no special precautions are taken, the intensity of light delivered to each individual optical fiber in a bundle (as well as the light directed to the reference photodiode) can differ strongly. If the alignment varies in time, even slightly, or there is a power fluctuation causing a change in the intensity of light, the variation will be different from fiber to fiber and from fibers to reference. This means that even slight changes in the relative position of the source and the multi-fiber bundle, or diffraction of the light beam by fluctuating air currents, will cause variations in signal intensity from the individual detectors, and the variations will not be correctable or normalizable.
Furthermore, it is often desirable to analyze a sample using broadband detection, i.e. detecting at a whole range or spectrum of wavelengths simultaneously. The use of mirrors or lenses to collect and focus light onto the input ends of multiple optical fibers can exacerbate the spatial and (if broadband sources are used) chromatic inhomogeneities of the source light distribution, thereby increasing the non-uniformities between the output of each of the individual fibers and the light directed to the reference photodiode. The Waters Corporation Model 2488 uses a grating to select only a single narrow wavelength band. However, if its design were modified to attempt to focus multiple wavelengths of light onto the input ends of the optical fibers the outputs of the individual fibers and light directed to the reference photodiode would be likely to also vary in spectral distribution.
To accurately compensate for the fluctuations in the intensity of the light from the incoherent light source, the light delivered to each optical fiber and the light directed to the reference diode must be substantially similar in intensity and spectral characteristics. “Similar” here refers to strictly proportional relationships between the profiles, both spatial and spectral, of the light delivered to each fiber and to the reference photodiode. It is also important that the proportionality constants relating these profiles remain stable so that they can be determined by calibration between measurements. For example, if one fiber transmits 1000 analog-to-digital-converter (ADC) counts at 254 nm and the other transmits 1100 ADC counts at 254 nm, then if the source lamp fluctuates upward to 1010 ADC counts as transmitted by the first, the second should transmit 1111 ADC counts.
If a broadband light source is used with the goal of broadband detection, the spectral characteristics of the light emanating from different areas of the source volume will also vary and chromatic aberrations will result from variations in the imaging of light of different wavelengths.
Alternatively, if the light source is a laser, the coherence properties of the laser light can cause only a limited number of waveguide propagation modes to be launched within the optical fibers. Because of this, the output is not spatially uniform but exhibits pronounced intensity fluctuations referred to as “speckle.”
Accordingly, there is a need in the art for a method for multiplexed optical detection wherein one light source is coupled to multiple optical fibers in such a way that the output of the optical fibers is substantially insensitive to movement of the fibers and substantially similar and homogeneous in terms of intensity and spectral profiles, and wherein changes in illumination of the multiple optical fibers by the light source results in uniform changes to the output intensity and spectral profiles of each output fiber.